This invention concerns a method for the determination of sialic acid, particularly lipid bound sialic acid, in whole blood which provides a number of advantages over existing methods involving blood plasma or sarum, including simplicity, cost, convenience, specificity and sensitivity.
Much work has been done which indicates that elevated sialic acid content in blood sera of a patient is an indication of the presence of cancer.
Thus, for example, U.S. Pat. No. 4,146,603 to Davidson et al. discloses and claims a fairly complex series of procedures whereby elevated sialic acid content is a determinant with respect to cancer specific determinations.
MacBeth and Bekesi, Cancer Res. 22:1170-1176 (1962) measured plasma glycoproteins and found galactose and mannose values were seen in breast cases without metastases. Kloppel et al., Proc. Natl. Acad. Sc, 74:3011--3013 (1977) reported 2.5-fold increases of serum sialic acid glycolipids in mice bearing transplantable mammary carcinomas and 2-fold increases in human carcinoma patients. The method involved column chromatographic separation of the gangliosides. A minimum of 1 ml whole blood was required Kloppel et al., Am. J. Vet. Res. 39:1377-1380 (1978) also reported increases of sialic acid in 92% of 24 dogs; however, a number of false positives were observed in dogs with other disorders. In leukemic AKR/J mice, Lengle, J. Natl. Cancer Inst. 62:1563-1567 (1979) found increased lipid bound sialic acid in their plasma and thymic lymphocytes. Lipid bound sialic acid levels were found increased in plasma and erythrocytes of humans bearing melanomas. Portoukalian et al., Biochem. Biophys. Res. Commun. 85:916-920 (1978). Chromatographic separation and purification on columns was followed by evaluation on chromatoplates. Silver et al., Cancer 41:1497-1499 (1978); Cancer Res. 39:5036-5042 (1979) have reported elevated serum sialic acid values in melanoma patients that were significantly related to the tumor burden. However, 36% of patients with observable tumors showed no elevated serum sialic acid. Hogan-Ryan et al., Br. J. Cancer 41:587-592 (1980) reporting on total bound serum sialic acid in patients with breast cancer found elevations that corresponded with tumor stage.
One specific method over which the present invention is an improvement is disclosed in the American Association for Cancer Research Annual Meeting PROCEEDINGS Vol 21, March 1980, as Abstract No. 728 by Katopdis et al. Briefly, this method requires that a 100 .mu.l plasma sample (reduced to 50 .mu.l ) be extracted with 6 ml of a chloroform/methanol mixture (2:1, volume to volume). The lipid extract is then partitioned with 0.2 of its volume of water. The aqueous phase is evaporated to dryness and the residue redissolved in water. The lipid bound sialic acid is then purified by trichloroacetic acid-phosphotungstic acid precipitation and, after the removal of the supernatant from the resultant precipitate, the precipitate is determined by the Svennerhelm and Miettien method (Svennerholm, Quantitative Estimation of Sialic Acid . . , Biochem. Siophys. Acta 24, pp. 604-611 (1957).
The other specific method over which the present invention is an improvement is disclosed in Katopodis and Stock, U.S. Pat. No. 4,342,567. This method is similar to the foregoing but requires only about 50 .mu.l of sample rather than the 100 .mu.l required by the prior art method. The drying step is eliminated and there is no use of trichloroacetic acid. Phosphotungstic acid is used alone. This prior method consists essentially of the following steps:
1. To a screw cap culture tube, 13.times.100 mm, add 150. distilled water with a 500.lambda. Hamiltion syringe. To this tube transfer a capillary pipette (Unopette, Becton-Dickinson 5841) with its content of 44.7.lambda. of plasma (or serum). Vortex the contents for 5 seconds. Transfer the tube to crushed ice.
2. Add to the tube 3.0 ml cold (4.degree.-5.degree. C.) 2:1 v/v mixture of chloroform and methanol and vortex the mixture for 10 seconds.
3. To this mixture add 0.5 ml cold distilled water, cap the tube and mix the contents by repeatedly inverting the tube for 30 seconds.
4. After centrifuging the tube 5 minutes at room temperature at 2500 rpm, transfer 1 ml of the upper layer into a culture tube like the one already used.
5. Add 50.lambda. phosphotungstic acid solution (1 g/ml) and after mixing let it stand at room temperature for 5 minutes.
6. Centrirage for 5 minutes at 2500 rpm and remove the supernatant by suction.
7. Add 1 ml water and vortex until the precipitate is in suspension without gross particles (about 1 minute).
8. Add 1 ml of the resorcinol reagent, mix and place the tube in boiling water for exactly 15 minutes.
9. Immediately after the 15 minutes, transfer the tube to an ice and water bath and leave for 10 minutes.
10. To the ice cold tube add 2 ml butyl acetate-n-butanol 85:15 v/v mixture at room temperature, vortex and centrifuge for 5 minutes at 2500 rpm.
11. Read the extracted blue color at 580 nm and the amount of lipid bound sialic acid (LSA) is determined by use of a standard curve developed from a standard sample of n-acetyl neuraminic acid (NANA) and use of this formula: EQU LSA (mg/100 ml plasma)=(x.multidot.100,000.lambda.)/(y.multidot.44.7.lambda..multidot.100 0)
x=.gamma. NANA read from standard curve for the sample PA0 y=l ml of supernatant .div. volume of entire supernatant
The preceding method suffers a number of disadvantages including the following: the need for a precisely defined 44.7.lambda. starting sample; the need for the sample to be in the form of plasma rather than whole blood; inconvenience; the increased labor, equipment, expense and likelihood of error associated with converting the sample from blood to plasma; the loss of lipid bound sialic acid during the tube inversion step creating reduced final values; incomplete precipitation of the lipid bound sialic acid with phosphotungstic acid, which is a particular problem when working with samples in which the amount exceeds normal values by only small amounts (e.g., early in cancer development); the 5 minutes' waiting time after phosphotungstic acid addition which limits the rapidity of the test and the undesirably high cost of the test.
Using the preceding method different laboratories have obtained results which vary widely. Table I sets forth results obtained by others and illustrates the variability obtained when samples from normal subjects were tested.
TABLE I ______________________________________ RESULTS OBTAINED BY DIFFERENT LABORATORIES USING THE METHOD OF U.S. Pat. No. 4,342,567 NORMAL SAMPLES RANGE MEAN UPPER LIMIT mgs % mgs % mgs % ______________________________________ 15.0-20.0 17.5 20.0 (1) 12.8-16.8 14.8 16.8 (2) 11.6-19.7 15.7 19.7 (3) 11.6-19.1 15.4 19.1 (4) 15.0-25.0 20.0 25.0 (5) 11.1-15.7 13.4 15.7 (6) 16.4-26.6 21.5 26.6 (7) NO INFO 15.3 NO INFO (8) NO INFO NO INFO 17.2 (9) 12.6-17.2 14.9 17.2 (10) 11.9-26.2 19.1 26.2 (11) 15.5-22.5 19.0 22.5 (12) 8.7-18.5 13.6 18.5 (13) 10.9-18.9 14.9 18.9 (14) 10.0-21.0 15.5 21.0 (15) MEAN- 12.3-20.6 16.4 18.2 ______________________________________ (1) KATOPODIS AND STOCK, U.S. Pat. No. 4,342,567 (2) CHEN SHUPAN et al., J. SHANGHAI MED. VOL. 6, 1983 (3) A. M. DNISTRIAN et al., CLINICAL CHEM. 27(10) 1981 (4) S. KAKARI et al., ANTICANCER RES. 4, Suppl. 1:36, 1984 (5) L. SANTAMARIA et al., MED. BIOLOGIE ENVIR. VOL. 12 1984 (6) A. M. DNISTRIAN et al., AACR VOL. 23, 609, 1982 (7) P. KOSMIDIS et al., ASCO, VOL. 2, C1, 1983 (8) D. MUNJAL et al., FED. PROC., VOL. 42(3), March 1983 (9) K. M. ERBIL et al., CL. CHEM. 29, VOL. 6(194), 1983 (10) CHEN SHUPAN et al., CHIN. J. OBSTET. & GYN. 18(4):23538 1983 (11) L. SALVAGNO et al., 13 INTL. CONG. OF CHEMO., 1983 (VIENNA) (12) L. SALVAGNO et al., I. OF CANCER RESEARCH, 1983 (13) A. K. BHARGAVA et al., ASCO, VOL. 6, No. 2, 1984 (14) S. KAKARI et al., INTL. MEETINGS, SALONICA, GREECE, 1982 (15) T. WUSTROW, GERMAN CANCER CONGRESS, 25/6 GL 1983